[BioC] limma, contrasts and affymetrix query

[Iain Gallagher]

Dear list

I wonder if someone could help me with some conceptual issues regarding limma, contrasts and analysis of an affymetrix study.

I have 8 samples (in duplicate) and 3 factors. The factors are O2 level (Norm, Hypo), Radiation (Yes/No) and Treatment (None, DMSO, Drug1, Drug2). 

The drugs were delivered to the cells using DMSO as a carrier. I am trying to use contrasts to examine the effect of the drug between two O2 levels taking into account the effect of DMSO:

TS <- paste(phenoData(rmaDataFiltered)$O2, phenoData(rmaDataFiltered)$Rtx, phenoData(rmaDataFiltered)$Treatment, sep=".")

TS <- factor(TS, levels=c("Hypo.No.None", "Hypo.No.DMSO", "Hypo.No.Drug1", "Hypo.No.Drug2",  "Hypo.Yes.None", "Hypo.Yes.DMSO", "Hypo.Yes.Drug1", "Hypo.Yes.Drug2",  "Norm.No.None", "Norm.No.DMSO", "Norm.No.Drug1", "Norm.No.Drug2", "Norm.Yes.None", "Norm.Yes.DMSO", "Norm.Yes.Drug1", "Norm.Yes.Drug2"))

design <- model.matrix(~0+TS)
colnames(design) <- levels(TS)
fit <- lmFit(rmaDataFiltered, design)

cont.matrix <- makeContrasts(HypoDrug1 = (Hypo.No.Drug1-Hypo.No.None)-(Hypo.No.DMSO-Hypo.No.None), NormDrug1 = (Norm.No.Drug1-Norm.No.None)-(Norm.No.DMSO-Norm.No.None), levels=design)

For these contrasts my thinking is as follows; I want to examine the effect of the drug versus untreated cells taking into account the effect of DMSO. So first I want to see the effect of DMSO on the untreated cells:

(Hypo.No.DMSO-Hypo.No.None) and (Norm.No.DMSO-Norm.No.None)

Then I want to remove this effect from the effect of the drug on untreated cells (i.e. create the difference of a difference):

HypoDrug1 = (Hypo.No.Drug1-Hypo.No.None)-(Hypo.No.DMSO-Hypo.No.None) (effect of drug - effect of DMSO)

and

NormDrug1 = (Norm.No.Drug1-Norm.No.None)-(Norm.No.DMSO-Norm.No.None) (effect of drug - effect of DMSO)

My question is am i going about this in the right way?

Thanks.

Iain